Liver Ex Situ Machine Perfusion Preservation: A Review of the Methodology and Results of Large Animal Studies and Clinical Trials

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1 REVIEW ARTICLE MARECKI ET AL. Liver Ex Situ Machine Perfusion Preservation: A Review of the Methodology and Results of Large Animal Studies and Clinical Trials Hazel Marecki, 1 Adel Bozorgzadeh, 1 Robert J. Porte, 2 Henri G. Leuvenink, 2 Korkut Uygun, 3 and Paulo N. Martins 1 1 Transplant Division, Department of Surgery, University of Massachusetts, Worcester, MA; 2 Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University Medical Center Groningen, Groningen, the Netherlands; and 3 Center of Engineering in Medicine, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA Ex vivo machine perfusion (MP) is a promising way to better preserve livers prior to transplantation. Currently, no methodology has a verified benefit over simple cold storage. Before becoming clinically feasible, MP requires validation in models that reliably predict human performance. Such a model has been found in porcine liver, whose physiological, anatomical, and immunological characteristics closely resemble the human liver. Since the 1930s, researchers have explored MP as preservation, but only recently have clinical trials been performed. Making this technology clinically available holds the promise of expanding the donor pool through more effective preservation of extended criteria donor (ECD) livers. MP promises to decrease delayed graft function, primary nonfunction, and biliary strictures, which are all common failure modes of transplanted ECD livers. Although hypothermic machine perfusion (HMP) has become the standard for kidney ex vivo preservation, the precise settings and clinical role for liver MP have not yet been established. In research, there are 2 schools of thought: normothermic machine perfusion, closely mimicking physiologic conditions, and HMP, to maximize preservation. Here, we review the literature for porcine ex vivo MP, with an aim to summarize perfusion settings and outcomes pertinent to the clinical establishment of MP. Liver Transplantation AASLD. Received October 4, 2016; accepted January 31, The shortage of organ transplantation donors is a worldwide problem. Although 5-year patient survival after orthotopic liver transplantation is over 70%, 1410 patients died on the waiting list in (1) The use of Abbreviation: AA, amino acids; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, adenosine triphosphate; CN, Custodiol-N; COR, controlled oxygenated rewarming; CS, cold storage; DBD, donation after brain death; DCD, donation after cardiac death; DGF, delayed graft function; ECD, extended criteria donor; ECLP, extra-corporeal liver perfusion; ECMO, extracorporeal membrane oxygenation; EGD, esophagogastroduodenoscopy; FABPL, fatty acid binding protein, liver; GGT, gamma-glutamyltransferase; GSH, glutathione; GST, glutathione S-transferase; HA, hepatic artery; HCT, hematocrit; HMGB1, high mobility group protein box 1; HMP, hypothermic machine perfusion; HOPE, hypothermic oxygenated machine perfusion; HTK, histidine tryptophan ketoglutarate; ICU, intensive care unit; IL, interleukin; IgM, immunoglobulin M; INR, international normalized ratio; IRI, ischemia/reperfusion injury; ITBL, ischemictype biliary lesion; KPS-1, kidney perfusion solution; LDH, lactate dehydrogenase; LFT, liver function test; mirna, microrna; MP, extended criteria donors (ECDs) is a promising way to expand the available donor pool. In particular, donation after cardiac death (DCD) liver grafts has become increasingly important as they have become a larger proportion of the donor pool, over 18% over the total donor pool in (1) DCD kidney grafts are used successfully for clinical transplantation. However, poorer outcomes have delayed the use of extrarenal DCD organ donations. DCD liver grafts, due to warm ischemic damage, carry higher risks for delayed graft function (DGF), primary nonfunction (PNF), and biliary complications following transplantation. (2) Biliary complications in DCD organs are a major source of morbidity, graft loss, and even mortality long term after liver transplantation. They are much more common in DCD grafts (20%-40% compared with 5% in grafts from brain-dead donors). (3) The most troublesome biliary complication is the ischemic-type biliary lesions (ITBLs), also called ischemic cholangiopathy. REVIEW ARTICLE 679

2 MARECKI ET AL. LIVER TRANSPLANTATION, May 2017 The risk of ischemic cholangiopathy in grafts from DCD donors is 10 times higher than for brain dead donors. (3) Eventually, up to 50%-65% of the patients with ITBLs will require a retransplantation or may die. (4,5) Most of DCD organs are discarded because of prolonged warm ischemia time (WIT; most centers decline when it is longer than 30 minutes). Because of poor results with DCD liver grafts after conventional cold storage (CS) preservation, interest in liver machine preservation was renewed. (6) Machine perfusion (MP) allows for the opportunity to flush waste products, to provide oxygen and nutrients, andtoreawakenlivermetabolicfunctionfollowingwarm ischemia (WI) and CS damage and could allow for measurement of graft viability ex vivo prior to transplantation. However, the simplicity and low cost of CS have kept it the standard of care for transplantation centers. Growing data support the use of MP preservation, but the variability of systems, techniques, settings, and costs have interfered with standardization and global use. Hypothermic machine perfusion (HMP) has been an attractive option to avoid further WIT and minimize graft metabolic requirements while providing oxygenation and metabolic support. Contrarily, some research supports the use of normothermic machine preservation (NMP) to most closely mimic the physiologic environment with oxygenation, nutrition, and full machine perfusion; mrna, messenger RNA; NAD1, nicotinamide adenenide dinucleotide; N/A, not available; NEMCO, normothermic extracorporeal membrane oxygenation; NMP, normothermic machine preservation; PCO 2, partial pressure of carbon dioxide; PEG, polyethylene glycol; PNF, primary nonfunction; PO 2, partial pressure of oxygen; PT, prothrombin time; PV, portal vein; PVP, peribiliary vascular plexus; RBC, red blood cells; RMP, rewarming machine preservation; RT-PCR, real-time polymerase chain reaction; SCS, simple cold storage; SEC, sinusoidal endothelial cell; SGOT, serum glutamic oxaloacetic transaminase; SNMP, subnormothermic machine preservation; Tbil, total bilirubin; TNF-a, tumor necrosis factor a; TPN, total parenteral nutrition; UW, University of Wisconsin; WI, warm ischemia; WIT, warm ischemia time. Address reprint requests to Paulo Martins, M.D., Ph.D., Division of Organ Transplantation, Department of Surgery, University of Massachusetts, 55 Lake Avenue North, Worcester, MA Telephone: ; FAX: ; paulo.martins@umassmemorial.org Copyright VC 2017 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /lt Potential conflict of interest: Nothing to report. metabolic support through both the portal vein (PV) and hepatic artery (HA). In addition, it has the advantage of testing if a liver is functional (bile production, lactate clearance) before transplantation, which cannot be tested during hypothermic perfusion preservation. Others have advocated for temperature profiles somewhere in-between. Subnormothermic machine preservation (SNMP) or rewarming machine preservation (RMP) represents the newest approaches in their attempts to maximally increase graft metabolism while minimizing reperfusion injury. Optimal temperature, mode of oxygenation, ideal perfusate, single versus dual perfusion, continuous versus pulsatile flow, and pressure and flow settings are debated, with little consensus as to ideal perfusion settings. Animal models have become crucially important for establishing transplantation methodology. Large animal research plays a crucial role in moving a therapy into a more complex in vivo system and evaluating the feasibility of a therapy. Before therapies are developed for clinical application, they require validation in models that are reliably predictive of their performance in humans. The physiological and anatomical characteristics of the pig closely resemble those of humans in many ways. Also, pigs are cheaper, and their use is more acceptable from the public s perspectives than nonhuman primates. (7) This review attempts to compile and summarize all independent studies of porcine machine liver perfusion in order to best comment upon machine settings, perfusate composition and additives, temperature profiles, as well as comparing the use of data collected during perfusions in the assessment of transplant viability. Historical Perspective In 1935, Alexis Carrel and Charles Lindbergh designed the first liver perfusion machine using pressure-controlled, oxygenated normothermic perfusate in a glass device. (8) Despite significant technical advances, no system has been proven to clinically significantly extend the length of ex vivo organ viability as compared with CS in preservation solutions such as University of Wisconsin (UW), histidine tryptophan ketoglutarate (HTK), and Celsior. The stilted progression of machine preservation is perhaps best explained by the history of kidney machine preservation. The perfusion preservation of kidneys first became part of clinical practice in the 1960s using a perfusate of blood and cryoprecipitated plasma. By the 1970s, HMP was used to preserve and 680 REVIEW ARTICLE

3 LIVER TRANSPLANTATION, Vol. 23, No. 5, 2017 MARECKI ET AL. transport kidneys. However, in 1980, the development of UW solution allowed for 72-hour kidney preservation by simple cold storage (SCS), and use of MP was discontinued. In recent years, the forced use of marginal donors resparked the use of HMP. An international multicenter trial of 672 paired kidneys found that HMP reduced the risk of DGF and decreased serum creatinine. (9) Now HMP is widely used clinically in the preservation of kidneys prior to transplantation. The use of MP for liver preservation has also been a long and complex journey, also beginning in the 1960s. After the first unsuccessful attempts of hypothermic perfusion of human livers by Starzl et al. in 1967, the clinical use and development of human liver perfusion devices did not move forward. (10) HMP, NMP, SNMP, and RMP have all been used successfully and shown to be safe in large animal models, with porcine models most commonly used. Because of the success of CS preservation in UW solution, there was no need to use machine preservation until the use of marginal livers became necessary due to increased discrepancy between demand and supply. In 2010, Guarrera et al. reported the first successful human liver transplantations following HMP preservation. (6) More recently, Dutkowski et al. have examined matched-case analyses of HMP versus CS with marked decrease of intrahepatic cholangiopathy, biliary complications, and improved 1-year graft survival. (11) Just in September 2016, Bral et al. published the first clinical normothermic machine perfusion (NMP) transplantation series, demonstrating feasibility and safety, but bringing to light questions of ideal methodology. (12) Porcine Model Large animal models provide an important step in establishing the feasibility of therapy in vivo. Porcine models were crucial to the development of clinical transplantation as pigs provide appropriate size and anatomy, as well as immunologic characteristics. In addition, pigs are cheaper and their use more publically acceptable than use of primates. Unlike dogs, and in line with human anatomy, pigs have no hepatic vein sphincters, and thus are ideal candidates for transplantation studies. (7) However, pigs have anatomical differences that require altering the transplant procedure significantly and are metabolically different and handle ischemic injury different than humans. Moreover, because the major complication that occurs in DCD liver transplant, ischemic cholangiopathy and consequent biliary stenosis, takes on the order of months to develop, ideally the recipient animal would be monitored for 3-6 months, which compounds the costs dramatically. (7) Still, most large animal models for machine preservation studies are in the porcine model, and a wealth of knowledge has been acquired through porcine perfusions. Perfusion Route Several different perfusion routes have been advocated for machine liver perfusion. PV-only perfusion is commonly used in rat liver perfusion. Arguments for HA perfusion include perhaps superior oxygen supply to the peribiliary vascular plexus. (13) Although it has been reported that the biliary ducts also have blood supply from the PV, (14) the biliary tract is mainly supplied with arterial blood through the peribiliary vascular plexus (PVP), and therefore, any injury to the PVP may contribute to the ischemic death of the biliary epithelial cells after transplantation. (15) The importance of HA perfusion is illustrated by the high incidence of biliary necrosis and strictures after HA thrombosis in patients after liver transplantation. (16) Additionally, during early reperfusion, the O 2 delivery is heavily dependent on arterial flow and as a result, dual perfusion is superior to portal perfusion in bile production as well as output of phospholipid, cholesterol, and bilirubin. (17,18) Combined arterial and portal MP of DCD porcine livers also results in a better preservation of the PVP and less arteriolonecrosis. (19) HA perfusion alone seems to be less beneficial than PV or dual perfusion. (20) All studies of porcine liver perfusion used either PV perfusion alone or dual perfusion through both the HA and PV (see Tables 1, 2, and 3). All NMP, SNMP, and RMP used dual perfusion circuits because these methods attempt to approximate physiologic conditions and maximize oxygenation. Figure 1 depicts a typical liver dual perfusion setup. Most HMP studies also used dual perfusion circuits with 3 studies out of 17 using PV perfusion only. PV-only studies stated goals such as simplifying MP or improving machine portability. No study seemed to argue that PV-only perfusion was superior or even comparable to the benefits of a dual perfusion system. Dual porcine MP through HA and PV appears to be more effective at alleviating reperfusion injury than either PV or HA alone. (17) The physiology of liver perfusion is notably complex, and an additional reason for dual perfusion is that it most closely mimics in vivo mechanics. For example, 1 study showed the principle of ex vivo flow REVIEW ARTICLE 681

4 Author (year) n WIT (minutes) CIT (hours) Perfusate Sch on et al. (42) (1993) Hellinger et al. (43) (1997) Butler et al. (34) (2002) 5 75 HPF31heparinized nonautologous blood versus 1 fructose, oleate, aa versus w/free rad scavengers 5 60 or 180 Krebs-Henseleit buffer, ph 7.4, 14% bolvine serum albumin, penicillin,streptomycin, heparin, aa 5 Heparinized porcine blood 1 CaCl2, NaHCO3 1 nutrition Imber et al. (35) (2002) St Peter et al. (22) (2002) Reddy et al. (23) (2005) 5 Short Heparinized whole blood versus with: CaCl2, NaHCO3, prostacyclin, taurochloric acid and TPN , flush Heparinized autologous blood 1 CaCl, NaHCO3, prostacyclin, taurocholate, nutrition, insulin or 4 Autologous heparinized blood 1 prostacyclin 1 taurochloric acid 1 nutrition Xu et al. (24) (2012) Boehnert et al. (30) (2013) 6 0 or 60 2 Porcine whole blood 1 plasma 1 hydrocortisone, insulin, penicillin, streptomycin, heparin Steen soln 1 cefazolin and heparin TABLE 1. Study Characteristics and Outcomes of Porcine MP Studies Using NMP Dual/PV Perfusion Perfusion Time, Hours Whole Blood Reperfusion Flow, ml/ minute Pressure, Pressure/Flow Controlled Flushes Temperature, 8C Oxygen End Points Outcome HA roller, PV gravity 3 Throughout (Hct 7) HA: , PV: 0.8-1/g liver HA: 50-75, PV: HA pressure, PV flow NMP (378C) Yes Histology, ALT, AST, LDH Metabolic support decreased ALT, AST, LDH release, metabolic support 1 NMP 1 neutralization radicals improve Dual circuit 3 No HA: 250, PV: 1180 Dual circuit 72 Throughout, hematorcrit monitored HA: 240, PV: 1750 Dual circuit 40 Throughout PV: , HA: HA: 70, PV: 5-10 HA: 90.3, PV: 7.22 PV: 5-8, HA: Pressure Flush with HTK for CS 48C (not NMP) HA: Flow, PV: Pressure Limited flow and pressure ranges NMP (378C) L Eurocollins NMP (398C) Yes, kpa 2 L cold Eurocollins Bile production, flow, oxygen consumption, creatinine kinase, LDH, transaminases, ph, electrolytes, complement/factor V production,, LFTs, bilirubin, base excess, O2 consumption NMP Yes Bile acid, Hbg,, ALT 1 hour WI plus 3 hours CS in HTK leads to viable organ and superior to 3 hours NMP in glutamine oxaloacetic transaminase and bile production, 3 hours WI nonviable organ Liver viability possible with NMP for at least 72 hours, synthetic function of urea and complement increased, AST and ALT decreased, little necrosis, some edema Bile production is better maintained by nutrient supplemented blood NMP and increased by supplementation with bile salts Dual circuit 24 Throughout PV: 1500, HA: 300 HA centrifugal, PV passive reservoir HA centrifugal, PV passive reservoir HA: 85-95, PV: 5-10 Flow 3 L cold Eurocollins 24 or 20 Throughout Physiologic Physiologic Pressure 3 L hyperosmolar citrate 4 C, then saline before NMP 4 Throughout (HCT 20) HA: , PV: HA: 70-80, PV: 5-8 Flow 2 L cold UW solution NMP Yes ALT, AST, LDH, bile production, factor V, glucose, NMP (398C) Yes Bile production, factor V (PT), base deficit, glucose use, AST, ALT, hyaluronic acid, NMP (398C) Yes AST, ALT, ATP, bile production,, O2 consumption HA and PV flow increase and bile production with NMP; AST/ALT/ LDH levels lower in NMP group, NMP mild vacuolization, nonviable organ after WIT and 24 hours CS 4 hours CS pre-nmp reduces bile production, factor V, and glucose, increases AST and ALT, base excess and endothelial injury (HyalA), hist. necrosis all noncorrectable NMP Liver enzyme, bile production, and pressure after WI improved by even 1 hour NMP, histologic changes of WI mostly reversed by 4 hours NMP Dual circuit 8 Yes HA: 365, PV: 950 HA: 60-80, PV: 4-8 Pressure Flush with cold UW NMP Yes ALT, oxygen extraction,, bile, HA angiography, AST, bilirubin, phospholipids, LDH NMP reduced ALT, minimized necrosis, improved bile production and flow, maintained O2 consumption, improved HA flow, reduced LDH as compared with CS

5 Author (year) n WIT (minutes) CIT (hours) Perfusate Liu et al. (26) (2014) 5 60 Porcine blood, heparin, calcium gluconate, sodium bicarb, cefotaxime, vancomycin, methylprednisolone Nassar et al. (27) (2015) 5 60 Whole blood versus 1 prostacycline or 1 adenosine Nassar et al. (36) 5 60 Whole blood 1 nutrition 1 insulin 1 vitamins Hoyer et al. (41) 5 and 6 18 Whole blood 1 gelofundin (Hct 20,378C) versus CN (gradually increased 8-208C) Liu et al. (44) 5 60 Steen soln versus steen plus RBC versus whole blood (WB) TABLE 1. Continued Dual/PV Perfusion Perfusion Time, Hours Whole Blood Reperfusion Flow, ml/ minute Pressure, Pressure/Flow Controlled Flushes Temperature, 8C Oxygen End Points Outcome HA centrifugal, PV continuous HA: 0.25/g liver, PV: 0.75/g Dual circuit 10 Throughout Total flow: /g liver Dual circuit hour simulation HA: 0.25/g liver, PV: 0.75/g HA: , PV: 3-5 HA: , PV: 7-12 HA: , PV: 3-5 Pressure HTK 2 L at 48C, prostacyclin through HA (vasodilator) Pressure (adj vasodil. HA, dec. flow PV) Pressure, flow adjusted with Flolan Dual circuit 3 Yes (180 minutes) PV: 0.8/g liver HA: 80 HA pressure, PV flow Dual circuit 10 hours Yes, 24 hours Flow decreased sooner without oxygen carriers HA: , PV: 3-5 Flush with HTK cold Flush with HTK cold Pressure Flush with HTK cold prior MP NMP (388C) Yes, above 350 PVF, HAF, AST, ALT, LDH, GGT, bicarb,, ph, liver weight NMP (398C) Yes AST, ALT, LDH, ALP, GGT,, hemodynamics NMP (38.58C) versus SNMP (218C) Yes ( ) CN COR versus NMP Yes (500 mm Hg) AST, ALT, LDH, GGT, bile production, bile bicarb, O2 consumption,, hemodynamics AST, ALT, vascular perfusion resistance, bile production, energy status, functional caspase 9, lipid peroxidation, NMP Yes AST, ALT, LDH, vascular resistance, bile composition and production, O 2 consumption, lactate, NMP group showed decreased LDH, GGT, AST, ALT and LDH compared with CS control with NMP group also showing significantly less ductal necrosis, PBGs injury, and arteriolonecrosis Prostacyclin > adenosine > just NMP in reducing AST, ALT, LDH and maintaining bile production and histologic architecture NMP resulted in lower AST and ALT, higher bile production, and better hepatocellular integrity and microcirculation on as compared with SNMP and CS After 18 hours CS, COR improves mitochondrial energy stores, has decreased AST/ALT, and displays superior mitochondrial as compared with NMP NMP with oxygen carriers (WB, or steen 1 RBC) decreases AST, ALT, and LDH and increases bile production. Whole blood perfusion produced best results in most parameters

6 Author (year) n WIT (minutes) CIT (hours) Perfusate TABLE 2. Study Characteristics and Outcomes of Porcine MP Studies Using SNMP and RMP Dual/PV perfusion Perfusion Time (hours) Whole Blood Reperfusion Flow (ml/ minute) Pressure () Pressure/Flow Controlled Flushes Temperature (8C) Oxygen End Points Outcome Gringeri et al. (29) (2012) Minor et al. (31) (2013) Knaak et al. (25) (2014) Izamis et al. (33) (2014) Obara et al. (32) (2013) Banan et al. (38) Furukori et al. (37) Morito et al. (40) Hagiwara et al. (39) 5 60 Celsior Dual input 6 Autologous Total flow: 0.5/ g liver 6 18 CN (at 8, 20, or gradually increased 8-20 degrees C) Steen, erythrocytes, sodium pyruvate, AA, calcium gluconate, insulin, cefazolin, metronidazole, heparin nutritive powder Williams Medium E 1 NaHCO3 1 heparin 1 insulin 1 dexamethasone UW gluconate1 dextran 3 0 or 4 Autologous heparinized blood (Hct 20-25) 1 Clinimix E4.25/5 and prostacyclin HA pulsatile, PV continuous HA centrifugal, PV gravity Monitored, not reported 1.5 Autologous PV: 0.80/g liver HA: 80 HA pressure, PV flow 8 Cellular perfusate throughout HA: , PV: 500 to 1100 Dual circuit 3 No HA: 0.16/g liver, PV: 0.50/g liver HA pulsatile, PV continuous 3 and 2 0 or 60 Modified UW HA centrifugal, PV gravity NR 60 2 Modified UW HA centrifugal, PV gravity 3 60 Whole blood 1 bicarb1 LMW dextran No PV: 0.22, HA: 0.06 HA pulsatile 4 Throughout HA: , PV: No PV: 0.22, HA: No 200 versus 60 inc to 200 Dual circuit 4 Throughout PV: 0.5/g liver HA: 0.20/g HA: 40 up to 70, PV: 3-5 (avoid over 8) PV: 5, HA: Flow Flush with 2 L Celsior Pressure, gravity (PV) Flow, adjusted for pressure PV flush HTK, HA with CN, pre blood: cold saline UW both bile duct and HA, flush out with saline prior to perfusion, dialysis 6-7 L lactated Ringer s, air into tube: major/minor SNP (28C) Yes AST, LDH, lactic acid, COR versus HMP/ SNP SNP (208C to 338C in 60 minutes) Outcome Flow None 48C-88C to238c gradual HA: 8015, PV: 611 Pressure Custodiol HTK at 4C, saline prior to perfusion, dialyzer Yes (500 mm Hg) SNP (208C-258C) Yes, 722 mm Hg 48C to388c in 120, 60, 30, and 20 minutes Outcome Flow None HMP versus rewarming to 238C Outcome Flow None 48C-88C to238c gradual HA: 30, PV: 10 Flow ECMO in situ 690 minutes 228, flush with Euro Collins cold versus no ECMO HMP versus SNMP (up to 228C-258C) Oxygen, AST, ALT, TNF-a, VR, high energy phosphates,, TLR4, Beclin-1 and ICAM-1, ECP and tissue ATP Yes, 95%-98% Flow, venous p02, AST,, PAS glycogen storage ph, 02 uptake,, bile production, ALT, U/S Yes Pressure change in HA, AST, ALT, LDH Yes AST, ALT, B- galactosidase, INR, bile production, hyaluronic acid, blood gases/oxygen extraction, LDH, ph, lactate, B- galactosidase Yes AST, ALT, LDH, hyaluronic acid, tissue ATP, TNF-a, IL-6, At 64 mg/l versus increased 13 to 64 Yes ( ) AST, ALT, HA pressures AST, LDH, hyaluronic acid, ph,, calculated pressure Lower AST, LDH and lactic acid with SNP versus CS after 60 minutes WI, also less histologic congestion, vacuolization, and necrosis after SNP than CS COR and SNP improved tissue energetics, COR reduced enzyme loss,tnf-a, peroxidation better than HMP/SNP, only COR increased bile production SNP using steen with RBC can recover homeostasis, metabolism, bile production, and oxygen consumption; comparable to pretransplant donor sample; no control group Embolisms cause flow degeneration, arterial embolism reduces bile flow, histological damage (and ALT) observed when both vessels damaged as meas. w/ contrast U/S Temperature-controlled rewarming MP drastically increased pressure variation (sign of graft function), and the release rate of LDH nearly to the level of a 0 minutes WIT liver Gradual rewardming of cold stored livers was safe: rewarming minutes led to better bile, INR and less IRI and biliary epithelial injury whereas gradual rewarming minimized hyaluronic acid (SEC damage) Gradual rewarming of livers after WIT resulted in decreased AST, ALT, hyaluronic acid and increased ATP stores as compared with HMP livers after WIT; no oxygen carrier used during rewarming Gradual rewarming of cold livers was most effective in minimizing AST, ALT and decreased HA pressure when also treated with gradual flow and oxygenation increase Combined use of in situ subnormothermic ECMP and subnormothernic preservation is superior in resuscitating DCD grafts

7 Author (year) n WIT (minutes) CIT (hours) Perfusate TABLE 3. Study Characteristics and Outcomes of Porcine MP Studies Using HMP Dual/PV perfusion Perfusion Time (hours) Whole Blood Reperfusion Flow (ml/ minute) Pressure () Pressure/Flow Controlled Flushes Temperature (8C) Oxygen End Points Outcome Jain et al. (42) (2005) Bessems et al. (47) (2006) van der Plaats et al. (48) (2006) Monbaliu et al. (43) (2007) Vekemans et al. (44) (2007) Monbaliu et al. (21) (2012) Obara et al. (45) (2012) Schlegel et al. (46) (2013) Dirkes et al. (50) (2013) Liu et al. (26) (2014) Li et al. (55) (2015) op den Dries et al. (19) (2014) 4 KPS-1 solution Dual circuit 24 Nonautologous 1 krebs-henseleit (20) 5 Celsior versus Polysol (colloid, impermeants, buffer, free rad scavengers, aa) 6 Belzer MP solution PV continuous HA pulsatile 6 KPS-1 (2 L recirculating) 5 KPS-1 (2L recirculating) KPS-1 HA and PV continuous HA: 0.1/g liver, PV: 0.3/g liver HA: 30, PV: PV only 24 No, krebs-henseleit PV: mm Hg 24 No HA: 80; PV: 350 Pressure 1 L Ringer s Lactate flush Flow 5 L Ringer s lactate flush Dual circuit 24 No PV: lim to 600 HA: 25, PV: 7 Pressure Prewash with HTK Dual circuit 24 No HF: PV: 1/g liver, LF: No PV: lim to 0.5/g liver or or 60 Modified UW Dual circuit 2-3 No HA: 0.19/g liver, PV: 0.26/g liver 8 0 or 60 6 UW 1 lactobionate, adenosine, deferoxamine, heparin, GSH versus 1 NADH PV only 1 Autologous (HCT 12-14) HF: PV , LF: PV HMP Yes O2 consumption,, bile production, ALT HMP (48C) mm Hg Ammonia clearance, urea production, bile production, hemodynamics, AST, ALT HMP maintained O2 consumption and decreased ALT production as compared with SCS Celsior caused more damage than polysol increasing AST, ALT, and intravascular resistance HA: 30, PV: 4 Pressure HMP (08C-48C) Yes 100 kpa LDH, PO2, Portable perfusion machine capable of maintaining liver 24 hours no significant damage HF: PV: 7, HA: 25; LF: PV: 3-5, HA: 20 HA: inc , dec 45, PV: lim to 7 versus inc 7-9 PV: flow, HA: pressure HA: pressure PV: lim pressure Prewash with HTK Prewash with 5 LHTK HMP None Bile outflow, VR, LDH, AST, FABPL, PO 2, PCO2, ph, HMP (48C-78C) Yes versus No Morphology and ATP perfusate HMP (48C-68C) None Q (PV&HA), calculated VR HA: 88, PV: 6 Flow HMP (48C-88C) Yes AST, ALT, LDH, minimum pressure of HA HF: PV: 8, LF: PV: 3 Pressure 208C saline pre WI, after WI 48C Celsior, no flush after CIT in control 3 Belzer MP soln PV, portable 20 No PV: 157 to 177 PV: 13 Pressure Pre-wash with 4L,48C HTK Belzer MP soln Dual circuit 4 Hct 7.3 PV lim to 0.5/g liver HA: 20, PV: 3 Pressure Pre-wash with 5L48C-68C HTK HMP (48C) Yes (>60kPa) versus No (<2kPa) NAD1, CO2, Cytochrome C, AST, HMGB1 (nuc. subcell injury),, TNF-a HMP Yes Blood gas, LDH, AST, and bilirubin,, VR HMP Yes ph, AST, FABPL, VR- HA, ATP, redoxact iron 5 30 Dual circuit 12 to 18 No HMP Yes AST, ALT, glucose, lactic acid, pyruvic acid, 9 30 Belzer MP soln (UW based with 100% 02) Dual circuit 4 Autologous HA: 200, PV: 480 HA: 30, PV: 5 Pressure Flush with HTK 48C HMP (108C) Yes, 30 during blood HA and PV flow, ATP, AST, LDH, bile production and composition, mrnas (BSEP,MDR3, CFTR, AE2),, caspase-3 No control--observations: HA VR higher than PV VR (constant) and HA VR decreases over first 6; : anoxic vacuoles after not before HMP, no bile production HMP increased ATP levels indicating better preservation of energy stores, oxygen was protective against anoxic vacuolization No control--demonstrated ex vivo flow competition--arterial pressure alters portal flow Normalized minimum HA pressure is a marker of WI damage and is alleviated by HMP 1 hour HOPE afrer 60 minutes WI positively affected cyt C, HMGB1,, AST release, reduced Kupffer cell activation (dec. TNFa), low pressure superior to high No control--feasibility of portable oxygen driven pump system Damage Index predictive of IRI depends on AST level and ph 18 hours HOPE delayed morphological change and apoptosis, and decreased release of AST/ALT with similar to baseline levels of glucose, lactic acid and pyruvic acid as compared to SCS HA flow and ATP content increased by HMP, AST and LDH lower in HMP group, improved after HMP (and less caspase-3)

8 MARECKI ET AL. LIVER TRANSPLANTATION, May 2017 FIG. 1. Typical liver MP setup. competition: arterial pressure alters portal flow. (21) For reasons of functional rejuvenation of liver tissue as well as providing consistent physiologic-mimicking perfusion, most researchers choose dual perfusion in porcine machine liver perfusion models. Normothermic Machine Perfusion Settings The tenant of NMP has long been the replication of physiologic parameters to minimize ischemia and maximize ex vivo metabolism. Hence, many porcine NMP trials use modified heparinized blood solutions or Steen solution plus erythrocytes as the perfusate. (21-28) Other studies used Celsior, (29) Steen, (30) Custodiol, (31) or UW gluconate (32) or modified nutritive Williams medium E (33) as perfusate. Custodiol is a modified version of the low viscosity HTK solution with additional iron chelators to inhibit cold-induced free radical medicated injury. Celsior is a colloid solution which aims to combine osmotic efficiency of UW (lactobionate and mannitol) with the buffering capability of HTK, while reducing tissue edema from hydrostatic pressure because Celsior macromolecules do not pass the cellular membrane. Liu et al. directly compared Steen solution versus Steen with erythrocytes versus whole blood with the result that aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) production was decreased and bile production was increased with improved in the groups with erythrocytes or whole blood, emphasizing the importance of an oxygen carrier in NMP. This study suggested and encouraged further study of acellular solutions such as Steen particularly given the demand to increase perfusate volume beyond the limit of available donor blood. However, the clear superiority of whole blood in NMP in this study exemplified the solutions limitations. All NMP studies used dual perfusion routes, mimicking physiology and perfused for a minimum of 3 hours up to as long as 24 hours. (26) Early studies aimed to lengthen liver storage up to 72 hours. (34) Other proponents of NMP claimed decreased ischemia/reperfusion injury (IRI) and resulted in superior bile production. (22,35) All NMP studies used oxygenators. Perfusion was controlled by either pressure of flow with flow being less popular in 5 out of 16 studies and a few studies using flow for HA and pressure for PV or vice versa. Goal pressures ranged from 50 to 100 mm Hg for the HA and from 3 to 10 for the PV. Flows ranged from 150 to 700 ml/minute for HA and from 950 to 1750 ml/minute for the PV, with a few studies reporting flow as a function of liver weight at HA ml/minute/g and PV as ml/minute/g (see Table 1). The benefits of NMP are nearly undisputed in the literature, but the addition of NMP into clinical practice is logistically complicated. Now that CS is the established standard preservation and that most centers lack perfusion machine availability, some period of CS in clinical practice is unavoidable. Four hours of CS prior to NMP results in statistically significant reduction in bile and factor V production, reduction of glucose stores, increased release of AST and ALT, and base excess and endothelial injury, as shown through increased hyaluronic acid as well as necrosis on, and none of these parameters were normalized if NMP was performed following CS. (23) The unavoidable period of CS has brought upon newer variations of NMP where the liver is perfused either at subnormothermic temperatures or the perfusate is gradually rewarmed from CS to normothermic temperatures (see Table 2). Initial perfusion at a cold temperature enables replenishment of cellular energy stores in order to reduce IRI upon rewarming. SNMP was shown to improve tissue energetics, peroxidation, and increased bile production (31) as well as better recover homeostasis, oxygen consumption, bile production, and. (25) However, others have found decreased bile production, increased AST/ALT, and worse using SNMP (218C) versus NMP (38.58C). (36) Temperature-controlled rewarming has been found to result in decreased release of LDH and superior mechanistic properties of the livers such as pressure curves. (32) Gradually warming warm 686 REVIEW ARTICLE

9 LIVER TRANSPLANTATION, Vol. 23, No. 5, 2017 MARECKI ET AL. ischemia treated livers to normothermic perfusion or subnormothermic perfusion was also shown to lower liver enzyme loss, increase adenosine triphosphate (ATP) stores, improve bile production, and improve as compared with hypothermic or subnormothernic perfusions. (31,37) Most recently, rewarming perfusion was shown to improve international normalized ratio (INR) and bile production as well as minimize sinusoidal endothelial cell injury and activation of Kupffer cells. However, extending the rewarming phase to 60 or 120 minutes rather than minutes decreased synthetic liver functions perhaps by increasing anaerobic respiration time. (38) Preliminary studies also suggest a period of in situ subnormothermic extracorporeal membrane oxygenation (ECMO) prior to rewarming to SNMP compounds the benefits of SNMP on resuscitation of livers after WIT. (39) Others found that gradual increase of perfusate flow and oxygen throughout the rewarming period minimized AST/ALT production. (40) Controlled oxygenated rewarming (COR) perfusion was also shown to be superior in recovering energy stores and minimizing hepatocellular and mitochondrial stores after 18 hours of CS as compared with NMP. (41) Hypothermic Machine Perfusion Settings In contrast to NMP settings, HMP settings are by definition not particularly physiologic, with perfusate temperature most commonly defined as 48C. Many porcine HMP studies used kidney perfusion solution (KPS-1), also known as UW solution of Belzer MP. (19,45-54) Bessems et al. was unique in comparing Celsior perfusate versus Polysol with the result that Celsior solution was more damaging than Polysol, increasing AST/ALT and intravascular resistance to a greater extent. (53) Polysol is a new low-viscosity preservation solution, containing amino acids, vitamins, potent buffers, and antioxidants to better support metabolism during HMP. One of the main components is polyethylene glycol (PEG) 35, a lowmolecular-weight colloid that does not increase viscosity. (53) Although experimental only, results for Polysol are promising, and it may be more widely used moving forward in ex vivo machine liver perfusion. All studies besides the exceptions noted in section perfusion routes used a dual circuit (see Table 3), and the most recent studies seem to agree that dual circuit HMP seems more beneficial. In many studies, the lengths of perfusion for HMP were much longer than NMP, up to 24 hours, as HMP is looked at as a potential means to extend the shelf-life of donated livers. Most studies since 2013 have focused on recovery of donor livers from WIT and have shorter perfusion times ranging from 1 to 4 hours. Most studies used pressure-controlled circuits with hepatic pressures (exception Monbaliu et al. with increasing pressures to 55 then 70 ) and portal pressures 3-13 with the majority limited to <7 mmhg (see Table 3). Studies in focused on defining the capabilities of HMP. HMP was shown to maintain superior oxygen consumption, when coupled with active oxygenation, and decrease ALT when compared with CS. (45) A portable perfusion machine was shown to maintain a liver for 24 hours with no significant histological damage. (53) The pattern of decreasing HA vascular resistance over the first 6 hours of perfusion was first noted by Monbaliu et al. Also noted in this study was the introduction of anoxic vacuoles by HMP, likely due to lack of oxygenation in this study, and the inability of their HMP livers to produce bile ex vivo. (46) Another study showed increased ATP stores after HMP, indicating better preservation of energy stores as well as use of an oxygenator directly protecting against anoxic vacuolization. (47) The modern focus of HMP has been primarily on hypothermic oxygenated machine perfusion (HOPE), and even low-flow oxygen has been shown to protect histologically against anoxic vacuolization. (54) Predicting Pretransplantation Viability A strong argument for the use of machine preservation is the added ability to assess viability of donor liver grafts prior to transplantation. Proposed outcome measures include flow and pressure characteristics and perfusate enzymes such as AST and LDH. The assessment of liver grafts is complicated by its unique dual blood supply, and the wide range and low flow portal system. Obara et al. found that hepatic arterial pressure, AST, and LDH correlated well with WIT during HMP. (48) Logically, oxygen consumption, complement/factor V production, bile production, and hyaluronic acid (marker of endothelial injury) have been used as outcomes and their levels correlated to damage found on. Other recent studies suggest the use of microrna (mirna) profiles of REVIEW ARTICLE 687

10 MARECKI ET AL. LIVER TRANSPLANTATION, May 2017 perfusate samples as indicators of WI injury and prediction of PNF. One mirna, mir-22, was correlated to transplantation survival outcome, but is believed to have low diagnostic potential. (55) Further work must be done to establish the best ex vivo markers of WI injury and reperfusion injury to determine the best predictors of good transplantation outcomes. When focusing on histologic changes caused by WI, it is important to perform reperfusion with whole blood because it magnifies IRI. Whole blood has all the key players of IRI, namely, the complement system, white blood cells, and platelets. One cannot expect to see all the changes associated with IRI if the liver is not perfused with blood. Op den Dries et al. showed after 30 minutes of WI and no CS, that HA flow and ATP content increased after HMP, whereas AST and LDH decreased significantly. (19) HMPtreated livers also showed improved and lower caspase 3 levels. This study was also significant for the use of messenger RNA (mrna) and the establishment of mrna profiles in the porcine model, which may be helpful in profiling livers damaged by WI. (19) After 60 minutes of WI and 5 hours of CS, Schlegel et al. found that the effects of 1 hour of hypothermic oxygenated portal perfusion on nicotinamide adenenide dinucleotide (NAD1), CO 2, cytochrome C, AST, high mobility group box 1 (HMGB1),, and tumor necrosis factor a (TNF-a) resulted in decreased cytochrome C, HMGB1, AST, and better with reduced Kupffer cell activiation (decreased TNF-a). (54) This study also compared low perfusion pressures with high perfusion pressures, showing that low pressure perfusion (3 ) was superior to high (8 ). Liu et al. studied ph, AST, fatty acid binding protein, liver (FABPL), ATP, and redox-activated iron after minutes of WI and no CS, attempting to create a damage index predictive of IRI. AST level and ph proved the most valuable predictors studied. (56) In a recent review by Verhoeven et al., bile production during NMP correlated well with early graft survival, as did bilirubin and phospholipid concentration, which are increased by NMP. (57) Impaired secretion of phospholipids after CS may result in a high bile salt/ phospholipid ratio known to be toxic to cholangiocytes and associated with the development of ITBLs. Hepatocyte injury is generally measured by release of AST, ALT, and LDH into the perfusate and is correlated with both WIT and poorer survival after transplantation. Traditionally energy status is recorded by ATP levels, and hyaluronic acid and thrombomodulin have become established markers for endothelial injury. Inflammatory markers such as TNF-a levels are shown to correlate positively on with Kupffer cell activation. Peribiliary epithelial and vascular injury is represented by arteriolosclerosis and changes in the luminal size of the PV branch, but these changes were only present after reperfusion. Lastly, a particular profile of mirna in the bile is associated with the later development of ITBLs. (55) Discussion Nearly a decade of ex vivo MP of livers has left modern medical science in much the place it started: what is the clinical role for ex vivo liver perfusion and how can it best be implemented? Simple CS in UW solution has become the gold standard such that any proposed perfusion must accept some period of interim CS prior to perfusion and that the perfusion must add some benefit beyond simple CS. At one point, the goal was to increase the length of the organ s viability as compared with CS, but with modern transportation and the organ donation system MP has not been rigorously shown to be effective in this regard. Modern goals of MP look toward restoring organ damage, for example extending donor criteria by alleviating damaging effects of WI in DCD livers. Additionally, modern perfusion systems look to test viability and predict dysfunction of donated livers prior to transplantation. Reviewing the successes and pitfalls of various MP techniques can show us where the greatest successes have been and where future studies should focus their energies. As early as 1970, Belzer et al. showed the promise of NMP in maintaining livers ex vivo by showing equivalent outcomes after 8-10 hours NMP as compared with immediate transplantation. (52) Sch on et al. showed that 4 hours of NMP could reverse, bile production, alpha glutathione S-transferase (GST), and AST levels after WI injury, and prevent the PNF seen within 24 hours of nonperfused livers. (28) However, many of these studies included no CS time. Brockmann et al. also showed improved survival with 5 or 20 hours of NMP, particularly in organs that had been severely ischemic (after 40 minutes of WI injury). (58) Of the porcine liver hypothermic machine preservation (HMP) studies, results have shown promising resuscitation of WI damaged livers, but the aspect of reperfusion injury remains largely untested. Obara et al. showed promise with the HA normalized 688 REVIEW ARTICLE

11 LIVER TRANSPLANTATION, Vol. 23, No. 5, 2017 MARECKI ET AL. pressure recovery to baseline with 2-3 hours of HMP. (48) Li et al. were only able to show slightly delayed morphologic change, apoptosis and release of AST/ALT with CS as compared with CS. (59) De Rougemont et al. showed HMP increased GSH stores and arterial pressure and HMP liver recipients survived where CS recipients did not. (60) In trials of transplantation after HMP (see Table 4), no benefit is seen as compared with CS (61) except in the resuscitation of unsalvageable livers after 60 minutes of WI (60) where transplants were viable only after HMP, and had improved glutathione (GSH) stores and arterial pressure. Many pig liver transplant studies showed survival rates as low as 33% (54) and 20%. (67) Most promising were livers with only 30 minutes of WI which HMP improved survival from 0% in CS to 75% in HMP with lower AST and LDH. (68) Although histologically and biochemically HMP-treated porcine livers were better preserved as compared with CS, the liver viability is still less than excellent in these preclinical trials. The clearest side-by-side demonstration of the effects of NMP, HMP, and RMP comes from a study by Shigeta et al. who studied livers after 60 minutes of WI and 2 hours of CS and compared n 5 3 orthotopic liver transplantations after 2 hours of either NMP, HMP, or RMP. In 24 hours, the study found all NMP and HMP livers suffered PNF with 0% survival, with 75% survival and improved in the RMP group. (69) Others have been able to show 100% survival after no WI and preservation at 218C RMP with total alleviation of postreperfusion syndrome, lower AST/ ALT, and less centrilobular necrosis. RMP transplants were perfused in UW gluconate solution with added branched-chain amino acids, with the SNP livers perfused using cell-free bovine-derived hemoglobin with hetastarch colloid. (70) With several animal trials showing liver MP to be safe and in many cases superior to simple CS, the first clinical trials have been recently reported and others are underway (see Table 5). Hypothermic machine preservation (HMP) has been the first clinically accepted method as preservation by HMP varies the least from SCS and was the first method to be successfully implemented in humans and reported by Guarrera et al. in (6) Implementation of HMP after a period of CS for ECD human livers was shown to improve ATP stores and results in increased bile, bilirubin, and bicarbonate production, but was not able to reduce hepatobiliary injury as measured by AST, ALT, LDH, and gamma-glutamyltransferase (GGT) as tested with NMP-simulated transplantation. (71) However, in another clinical trial by Dutkowski et al. performed in 2 European hospitals, DCD livers treated with oxygenated HMP prior to transplantation had reduced ALT peak, cholangiopathy, and biliary complications and improved 1-year graft survival compared with SCS-treated DCD grafts and decreased necessity of retransplantation (18% SCS to 0% HMP). (11) The first human liver was transplanted after resuscitation with NMP in 2015 (72) with subsequent studies summarizing their results. (73) The first clinical trial of NMP feasibility in transplantation by Ravikumar et al. took place in Oxford, UK, and showed similar 30-day graft survival and lower peak AST in the first 7 days as compared with CS. (73) The first North American NMP clinical trial was carried out in Toronto by Selzner et al. using Steen solution as perfusate with no difference detected in graft function, proving safety and comparable outcomes to CS. (75) Recently, another Canadian single-center study in Edmonton by Bral et al. with similar study design of the Oxford trial showed the feasibility of 10 human DCD and donation after brain death (DBD) livers treated with NMP for a mean of 11.5 hours prior to transplantation, with a small decrease in 6-month graft survival in NMPtreated livers (80%) versus SCS livers (100%), demonstrating the technical difficulty of implementing liver NMP clinically. (12) These unexpected results compared to the UK trial was speculated to be related to the higher proportion of DCD livers (44% Edmonton trial versus 20% for UK or Toronto trials) or the prolonged cold ischemia time (median, 2 hours 47 minutes) due to uncommonly complex back-table reconstructions, and HTK donor flush in the Edmonton trial. Additionally, the superiority of COR and SNMP in porcine livers sparked the first successful application or COR in clinical liver transplantation. (41,79) Rewarming therapies, the newest methodology in machine preservation, may become the ultimate compromise with their ability to capture advantages of relative convenience as compared with NMP, longevity of storage comparable to HMP or SCS, optimal resuscitation in predictive animal models, and predictive ex vivo assessment prior to transplantation. In conclusion, the porcine model has provided a stepping stone and a wealth of iterative learning about ex vivo liver MP preservation. With physiological, anatomical, and immunological properties similar to humans, porcine livers have allowed the modeling of WI and IRI as well as establishing markers predictive of posttransplantation outcomes. With SNP and REVIEW ARTICLE 689

12 Author/year N WIT (minutes) CIT (hours) Perfusate TABLE 4. Study Characteristics and Outcomes of Porcine MP Preservation Followed by Orthotopic Porcine Liver Transplantation Length of Perfusion (hours) Perfusion Route Flow Pressure Pressure/Flow Controlled Treatments (Extra Substances) Hypothermic or Normothermic Oxygen Follow-up End Points Outcome Belzer et al. (51) (1970) Nishitai et al. (62) (2001) Sch on et al. (28) (2001) Foley et al. (18) (2003) Guarrera et al. (63) (2005) Brockmann et al. (58) (2009) de Rougemont et al. (60) (2009) Vekemans et al. (54) (2009) Monbaliu et al. (64) (2009) Fondevila et al. (67) (2011) Fondevila et al. (65) (2012) 4 or 5 N/A N/A Porcine plasma with ACD soln cryoprecipitated 5 N/A N/A Baboon blood with ringer s lactate to Hct 30% 6 0 or 60 minutes N/A 2L pig heparinized blood11l electrolyte soln 1 diasylate 8 or 24 Nonpulsitile PV, pulsitile HA 5 circulations for a max total 6 Dual ECLP in vivo baboons ml/minute/ g liver Total 1 ml/minute (HA:PV 1:4) 4 Dual HA: 150 ml/minute, PV: 250 ml/minute 4 N/A 120 minutes Bile acid Dual versus single (PV) 548 ml/minute single versus 727 ml/ minute double HA: 60/40, PV: 5-8 HA <200 mm Hg, PV<60 HA: 100 mm Hg, PV: PV: 8-12 mm Hg, HA 3 N/A N/A Vasosol soln 12 Dual circuit 0.7 ml/g/minute PV: 3-5 mm Hg, HA: to 7 40 or 60 N/A Heparinized donor blood1nahco31 prostacyclin1 taurochlorate 1 aa hr Starch-free UW solution with lactobionate, adenosine, deferoxamine, heparin, GSH 3 N/A N/A KPS-1 (EKPS-1) versus Aquix RS-I hour (postperfusion) Ringers1 streptokinase 1 epoprostenol hours Ringers, Voluven, NaHCO3, heparinized blood HA: Pressure, PV: Flow Flow Brief flush with heparinized Ringers Pressure Dialyzed perfusate Pressure N livers transplanted, N studied ex vivo Flow Flush with UW solution NMP (88C-108C) Yes 5-34 days Alkaline phosphatase, bilirubin, SGOT Biological HA: 45-60, PV: PO 2 maintained Few days LDH, ALT, PBMC flow cytometry,, porcine IgM and G, Hb NMP (378C) Yes 7 days Bile production,, alpha GST, INR, hyualuronic acid, AST, ALT NMP Extracorporeal, dual: 1.8 ml/minute/100 g versus single: day Bile production, O 2 consumption, cholesterol/phos. choline ratio NMP of 8-10 hours was functionally equivilant to immediate transplant in survival and ECLP has no serious longterm influence on cellular immunity 4 hours NMP versus CS reverses WI injury: decreased alpha GST, AST, improves bile&hist. prevents PNF seen w/in 24 hours in CS Dual circuit slightly superior to single circuits in increasing bile productions, but ex vivo production<in vivo regardless HMP (38C-58C) No 5 days AST and bilirubin AST and Tbil similar between CS and HMP groups, HMP safe and reliable; 1 arterial thrombosis in CS, no proven benefit 5 or 20 HA pump, PV reservoir Physiological Pressure NMP Yes 5 days Bile production, base excess, PVR, enzymes,, caspase3 assay 1 Single PV ml/minute 8 mbar Pressure Flush with 1L heparinized saline, then 111 L Celsior 4 Dual circuit PV 0.25 ml/g liver, HA unlisted PV: 3-5, HA: 20 1 warm flush Both HA and PV N/A N/A By gravity 48C HTK after Ringer s preflush 4 Dual perfusion PV: 8, HA: 60/40 mm Hg hours UW-MP 4 Dual centrifugal HA: , PV: PV: 4, HA: 30/ 20 Pressure Flush HTK HMP (58C-88C) Yes ( mm Hg) Pressure NEMCO 60 minutes HMP Yes 7 days Bile flow, AST, ALT, bilirubin, INR, hematocrit, lactate, platelets,, GSH, ATP 3 days Survival,, AST, TNF-a, hyaluronic acid Normo flush No 60 hours Survival, AST, PT, and factor V, TNF-a, redoxactive iron, bile flow and composition, NMP Extracorporeal membrane Pressure HMP (48C) HILITE membrane oxygenator 5 days Bile production, TNFa, IL6,, PNF, death 5 days Survival,, quick PT, PVF, HAF, AST, ph, PO2 No difference at 5 hours, 20 hours: improved survival with NMP versus CS, particularly after 40 minutes WI, no survivors 60 minutes WI HMP increases GSH stores and arterial pressure; ischemic livers in CS killed recipient, with HMP did not HMP survival 33%, reduced TNFa, hyaluronic acid clearance lower in EKPS-1 (less endothelial cell dysfunction) Drastically improved function and survival with warm flush, lower TNF-a, and redox-active iron NEMCO superior to CS, NMP with NEMCO superior to NEMCO in damaged grafts Improved survival (20 versus 0%) with HMP, increase TNF-a and endothelial and Kupffer cell injury with HMP